Neuroglobin (Ngb) is a recently discovered tissue globin found in the vertebrate brain. Initial study suggests that NGB is protective against hypoxia-ischemic insults. However, the mechanisms underlie Ngb neuroprotection remain to be defined. [unreadable] [unreadable] Our over all hypothesis is that after hypoxia/ischemia, elevated Ngb modulates neuron survival by (a) facilitating oxygen diffusion thus improving mitochondrial respiration and function, (b) scavenges neuronal nitric oxide (NO) thus reducing the formation of reactive nitrogen species (RNS) and reactive oxygen species (ROS), and (c) down-regulating cell death signaling and diminishing glutamate release. [unreadable] [unreadable] In Aim 1, we will examine the role of Ngb in regulating mitochondria respiration and neuronal death signaling after hypoxia/reoxygenation (H/R) in vitro. We hypothesize that Ngb is upregulated after H/R in mouse cerebral cortical neurons and over-expressing Ngb improves mitochondria respiration and down-regulates neuronal death signaling such as cytochrome c release and caspase activation. In Aim 2, we will document the specific role of NO-scavenging by Ngb after H/R in vitro. Ngb will be overexpressed in neurons isolated from nNOS knockout mice and matching wild-type mice. Changes of RNS and ROS formation, mitochondrial respiration, neuronal death signaling and glutamate release will be examined and compared. In Aim 3, we will compare Ngb overexpressing transgenic mice with wild type mice after focal cerebral ischemia in vivo. Alterations of neurological outcomes, ATP levels, RNS and ROS formations, cell death signaling, and glutamate release will be measured. To further dissect the role of Ngb in regulating neuronal NO-mediated brain damage, Ngb transgenic mice will be crossed with nNOS knockout mice. [unreadable] [unreadable] These proposed experiments should provide new insight of how Ngb protect neurons from hypoxia/ischemia and may ultimately lead to novel therapeutic strategies for the treatment of stroke. [unreadable] [unreadable]